DE69432179T2 - A FORMULATION OF THE COagulation Factor VIII - Google Patents

Abstract

PCT No. PCT/SE94/00297 Sec. 371 Date Dec. 21, 1995 Sec. 102(e) Date Dec. 21, 1995 PCT Filed Mar. 31, 1994 PCT Pub. No. WO95/01804 PCT Pub. Date Jan. 19, 1995A pharmaceutical formulation for subcutaneous, intramuscular or intradermal administration comprising recombinant coagulation factor VIII and use thereof for manufacture of a medicament for treating haemophilia is provided. The formulation comprises a highly purified recombinant coagulation factor VIII in a concentration of at least 1000 IU/ml, which gives surprisingly high levels of active factor VIII in the blood stream after subcutaneous, intramuscular or intradermal administration. The formulation is intended for treatment of haemophilia by subcutaneous, intramuscular or intradermal administration. The recombinant factor VIII is preferably a deletion derivative thereof, which can be used for the manufacture of a medicament for subcutaneous administration.

Description

The present invention relates to a pharmaceutical formulation for subcutaneous, intramuscular or intradermal administration comprising recombinant coagulation factor VIII and the use thereof for the manufacture of a medicament for the treatment of haemophilia. The formulation comprises a highly purified recombinant coagulation factor VIII in a concentration of at least 1000 IU/ml, which results in surprisingly high levels of active factor VIII in the bloodstream after subcutaneous, intramuscular or intradermal administration. The formulation is intended for the treatment of haemophilia by subcutaneous, intramuscular or intradermal administration. The recombinant factor VIII is preferably a deletion derivative thereof which can be used for the manufacture of a medicament for subcutaneous administration.

Background of the invention

Haemophilia is a hereditary disease that has been known for centuries, but it has only been possible in the last three decades to differentiate between the different forms of haemophilia A, haemophilia B and haemophilia C. Haemophilia A is the most common form. It affects only males, with an incidence of one or two individuals per 10,000 live males. The disease is caused by a severely reduced level or absence of biologically active coagulation factor VIII (antihaemophilic factor), which is a protein normally present in plasma. The clinical manifestation of haemophilia A is a severe bleeding tendency, and before treatment with factor VIII concentrates was introduced, the average age of these patients was less than 20 years. Concentrates of factor VIII obtained from plasma have been available for about three decades. This has significantly improved the situation for the treatment of haemophilia patients and given them the opportunity to live a normal life.

Therapeutic factor VIII concentrates have been produced to date by fractionation of plasma. However, methods for producing factor VIII in cell culture using recombinant DNA techniques are now available, as reported, for example, in J. Gitschier et al., Nature 312, pages 330-37, 1984 and EP-A-160 457.

Factor VIII concentrates derived from human plasma contain several fragmented, fully active forms of factor VIII (Andersson et al., Proc. Natl. Acad. Bei. USA, Volume 83, pages 2979-83, May 1986). The smallest active form has a molecular weight of 170 kDa and consists of two chains of 90 kDa and 80 kDa held together by a metal ion bridge. Reference is made here to EP-A-197 901.

Kabi Pharmacia has developed a recombinant factor VIII product that corresponds to the 170 kDa form of plasma factor VIII in therapeutic factor VIII concentrates. The truncated recombinant factor VIII molecule is called r-VIII SQ and is produced by Chinese hamster ovary (CHO) cells in a cell culture process in serum-free medium.

The structure and biochemistry of the recombinant factor VIII products were generally described by Kaufman in Tibtech, volume 9, 1991 and Hematology, 63, pages 155-65, 1991. The structure and biochemistry of r-VIII SQ were described in WO-A-91/09122.

Large proteins are usually given intravenously so that the drug is available directly in the bloodstream. However, it would be advantageous if a drug could be given subcutaneously, intramuscularly or intradermally as these administration routes are much easier for the patient to handle. This is especially true if the drug must be taken regularly throughout life and treatment should begin early, when the patient is a child.

However, a drug containing a very large and labile molecule, such as coagulation factor VIII of 170 to 300 kDa, usually has very low bioavailability when given subcutaneously, intramuscularly or intradermally, because of insufficient absorption and severe degradation. To our knowledge, the only coagulation factor protein that has been administered by subcutaneous injection is factor IX (90 kDa).

All Factor VIII preparations currently available on the market are manufactured as a formulation for intravenous administration and are stabilized with human serum albumin.

Description of the invention

To our great surprise, we have found that factor VIII, which is a very sensitive protein, can be given subcutaneously and, contrary to all previous experience, we have obtained acceptable absorption and high levels of active factor VIII protein in the blood.

We have thus developed a formulation that makes it possible to administer Factor VIII subcutaneously, intramuscularly or intradermally and which offers a great advantage for all patients who require Factor VIII.

Recombinant factor VIII SQ is indicated for the treatment of classical hemophilia.

The half-life for r-VIII SQ in humans is approximately 12 hours when injected intravenously.

For prophylactic treatment, 15 to 25 IU/kg body weight of factor VIII is given three times a week. An intravenous injection usually contains 5 to 10 ml.

An injection given subcutaneously contains between 0.05 and 1 ml, and the concentration of factor VIII must therefore be very high in such a formulation.

This can be achieved, for example, with our highly purified recombinant factor VIII.

The inventive idea is thus a combination of the discovery that factor VIII can be absorbed into the bloodstream when given as a subcutaneous, intramuscular or intradermal pharmaceutical formulation and that it is possible to prepare a formulation comprising the required high concentration of factor VIII for this purpose.

The present invention relates to a pharmaceutical formulation for subcutaneous, intramuscular or intradermal administration, which contains highly purified recombinant coagulation factor VIII in a concentration of at least 1000 IU/ml, the formulation providing a therapeutic level of Factor VIII activity in the blood following administration.

The composition is preferably administered subcutaneously.

The factor VIII activity in the formulation is at least 1000 IU/ml, preferably more than 1500 IU/ml and most preferably from 5000 to 100 000 IU/ml.

The volume dispensed is suitably 0.1 to 2 ml, preferably 0.25 to 1.5 ml, and more preferably 0.5 to 1 ml. The volume may also be 0.1 to 1 ml.

Factor VIII is recombinant and can be present either in its full length form or, preferably, as a deletion derivative thereof. In particular, the deletion derivative is recombinant factor VIII SQ (r-VIII SQ). Deletion derivative here means coagulation factor VIII lacking all or part of the B domain. In addition, the r-VIII SQ molecule can be chemically modified, e.g., by pegylation, covalently linked to carbohydrates or polypeptides, to improve the stability of the molecule in vivo.

The factor VIII we use is highly purified, i.e. it has a specific activity of more than 5000 IU/mg protein, even more than 12 000 IU/mg, and is preferably stabilized without the addition of albumin.

The formulation may also comprise sodium or potassium chloride, preferably in an amount of more than 0.1 M.

Calcium (or other divalent metal ions) is necessary to maintain the association of the heavy and light chains of factor VIII. It is added here as calcium chloride (CaCl2), but other salts such as calcium gluconate, calcium glubionate or calcium gluceptate may also be used. The composition preferably comprises calcium chloride or calcium gluconate in an amount of more than 0.5 mM.

An amino acid is preferably used to buffer the system and it also protects the protein in the amorphous phase when the formulation is freeze-dried. A suitable buffer would be L-histidine, lysine and/or arginine.

L-histidine was selected mainly because of the good buffering capacity of L-histidine around pH 7.

The wording could include

(i) at least 1500 IU/ml of a deletion derivative of recombinant factor VIII,

(ii) at least 0.01 mg/ml of a polyoxyethylene sorbitan fatty acid ester,

(iii) sodium chloride, preferably in an amount of more than 0,1 M,

iv) calcium salt, such as calcium chloride or calcium gluconate, preferably in an amount of more than 0.5 mM,

(v) an amino acid such as L-histidine in an amount exceeding 1 mM.

A non-ionic surfactant may also be present in the formulation and is then preferably selected from block copolymers such as a poloxamer, or a polyoxyethylene sorbitan fatty acid ester such as polyoxyethylene (20) sorbitan monolaurate or polyoxyethylene (20) sorbitan monooleate. The nonionic surfactant, if used, should preferably be present in an amount above the critical micelle concentration (CMC). See Wan and Lee, Journal of Pharm Sci, 63, page 136,1974.

The polyoxyethylene sorbitan fatty acid ester is preferably used in an amount of at least 0.01 mg/ml.

Mono- or disaccharides or sugar alcohols, preferably sucrose, could be added to this formulation. Antioxidants such as glutathione, acetylcysteine, tocopherol, methionine, EDTA, citric acid, butylhydroxytoluene and/or butylhydroxyanisole could also be added. Furthermore, preservatives such as benzyl alcohol, phenol, sorbic acid, parabens and chlorocresol could be added.

The formulation preferably comprises L-histidine and sucrose. The ratio of sodium chloride to L-histidine and sucrose in the freeze-drying composition is suitably more than 1:1 (w:w), preferably more than 2 : 1 (w : w).

The formulation could be in a dried form, preferably freeze-dried. The dried product is reconstituted with sterile water for injection or a pharmaceutically acceptable buffer solution or mixed or reconstituted with an aqueous solution containing absorption enhancers or protease inhibitors prior to administration.

The claimed formulation may also be a stable aqueous solution ready for administration. It may also be a dispersion, e.g. a suspension, a liposomal formulation or an emulsion.

The formulation could be stored in a reduced oxygen environment as disclosed in the co-pending patent application PCT/SE 94/00265.

Absorption enhancers or protease inhibitors could be added. Examples of suitable absorption enhancers are phospholipids, fatty acids, bile salts, salicylates and EDTA. Examples of suitable protease inhibitors are aprotinin and EDTA.

The claimed formulation can be prepared by mixing factor VIII with a non-ionic surfactant in an aqueous solution, preferably together with an amino acid such as L-histidine, sodium salt, sucrose and a calcium salt or by eluting factor VIII from the last purification step with a buffer containing a non-ionic surfactant in an aqueous solution, preferably together with sodium salt, sucrose, calcium salt and an amino acid such as L-histidine.

The invention also relates to the use of the claimed formulation for the manufacture of a medicament for subcutaneous, intramuscular or intradermal administration for the treatment of haemophilia, preferably the use of a deletion derivative of recombinant factor VIII for the manufacture of a medicament for subcutaneous administration. The medicament may be in a stable aqueous solution or dispersion or freeze-dried. It also relates to a method for the treatment of haemophilia by subcutaneous, intramuscular or intradermal administration of the claimed formulation.

The data presented in the examples show that r-VIII SQ can be injected subcutaneously and returned intravenously in an active form in vivo. This is a very surprising discovery, since no such formulations were known before.

The protection is not limited to a composition that falls under these examples.

EXPERIMENTAL Materials and methods

The production of recombinant factor VIII SQ (r-VIII SQ) was carried out essentially as described in patent WO-A-91/09122, examples 1-3. A DHFR-deficient CHO cell line (DG44 N.Y.) was electroporated with an expression vector containing the r-VIII SQ gene and an expression vector containing the dihydrofolate reductase gene. After selection on selective media, surviving colonies were amplified by culturing in stepwise increasing amounts of methotrexate. The supernatant from the resulting colonies was individually screened for factor VIII activity. A clone from the production was selected and this was subsequently adapted to growth in a serum-free suspension in a defined medium, and finally a large-scale fermentation process was developed. Supernatant is collected at certain intervals and further purified as described below.

The pH of the clarified conditioned medium was adjusted and applied to an S-Sepharose FF column. After washing, factor VIII was eluted with a saline buffer containing 5 mM CaCl2.

Immunoadsorption was performed on an immunoaffinity resin, where the ligand was a monoclonal antibody (8A4) directed against the heavy chain of factor VIII. Before loading onto the column, the S-eluate was treated with 0.3% TNBP and 1% octoxynol 9.

The column was equilibrated, washed, and factor VIII was eluted with a buffer containing 0.05 M CaCl2 and 50% ethylene glycol.

The mAb eluate was loaded onto a Q-Sepharose FF column equilibrated with the elution buffer from the immunoaffinity step. After washing, Factor VIII was eluted with 0.05 M L-histidine, 0.6 M sodium chloride, 4 mM calcium chloride and pH 6.8.

The Q-eluate was applied to a gel filtration column (Superdex 200 p. g.). Equilibration and elution were performed with a buffer containing L-histidine, sodium chloride and calcium chloride. The protein peak was collected and the solution was formulated before freeze-drying.

The material of r-VIII SQ was obtained from the final purification step. The Factor VIII activity and the concentration of the inactive components were adjusted by dilution with an appropriate buffer containing polyethylene glycol (PEG). The solution was then sterile filtered (0.22 µm), partitioned and freeze dried.

EXAMPLE 1

Recombinant factor VIII was prepared according to the procedure described in Experimental Section.

The freeze-dried composition containing r-VIII SQ was as follows per vial, which was reconstituted in 4 ml of sterile water for injection:

Composition per bottle:

L-Histidine, mg 31.0

Sodium chloride, mg 70.1

Calcium chloride (2H2 O), mg 2.35

Polyethylene glycol (PEG 4000), mg 4.0

Polyoxyethylene (20) sorbitan monooleate

(Tween 80®), mg 1

VIII:C filled, IU/vial 4400

VIII:C in reconstituted solution, IU/ml 1060

Male albino mice weighing approximately 30 g and belonging to the NMRI, SPF strain were injected subcutaneously into the neck with the reconstituted r-VIII SQ solution.

The injected volume was 9.4 ml/kg at the dose level of 10,000 IU/kg and 47 ml/kg at the higher dose level of 50,000 IU/kg, five times more. In the placebo treatment, saline solution, 9.4 ml/kg. 3 to 5 minutes before taking blood samples, mice were anesthetized intraperitoneally with Mebumal® (pentobarbital) vet. 60 mg/ml. The volume injected was 9.4 ml/kg, ie approximately 0.3 ml/mouse. Under anesthesia, 0.45 ml of blood was withdrawn from the vena cava into plastic syringes containing 0.05 ml of 0.13 M sodium citrate. Plasma was then prepared from the withdrawn blood by centrifugation (8800 g for 7 minutes) and stored frozen at -70ºC in Cryoflex plastic tubes until the time of determining factor VIII activity.

Results TABLE 1

VIII:C in plasma from mice receiving r-VIII SQ, 10,000 IU/kg body weight, subcutaneously.

TABLE 2

Dose-response in mice receiving subcutaneous r-VIII SQ.

Dose: administered dose of r-VIII SQ (IU/kg).

Response: VIII:C in plasma (IU/ml), 1.5 hours after subcutaneous administration.

Results

1. The change in plasma VIII:C with time following a subcutaneous dose of r-VIII SQ, 10,000 IU/kg, shows the typical pattern for a drug absorbed from a subcutaneous depot (see Table 1). The maximum plasma VIII:C level is seen at approximately 1.5 hours after administration (see Table 1).

2. Absorption of r-VIII SQ from a subcutaneous depot is further verified by the increase in maximum concentration seen when the dose is increased 5-fold. No significant effect on the obtained plasma level of VIII:C was observed when the subcutaneous injection volume was changed from 9.4 to 47 ml/kg while the dose of r-VIII SQ was kept constant. Furthermore, the VIII:C obtained in plasma was essentially independent of the osmolality of the administered solution.

3. The bioavailability of r-VIII SQ after subcutaneous administration to the mouse was approximately 10% of the bioavailability after intravenous administration. The bioavailability was calculated from the area under the activity (VIII:C)-time curve.

EXAMPLE 2

Plasma-derived factor VIII was used.

The composition of the frozen solution containing plasma-derived factor VIII was as follows:

Composition per ml:

L-Histidine, mg 7.8

Sodium chloride, mg 35.1

Calcium chloride (2H2 O), mg 0.59

Polyethylene glycol (PEG 4000), mg 1.0

human albumin, mg 10.0

VIII:C, IU/ml 250

Male albino mice weighing approximately 30 g and belonging to the NMRI, SPF strain were injected subcutaneously into the neck with the reconstituted solution of factor VIII.

The volume injected at the dose level of 10,000 IU/kg was approximately 40 ml/kg. For placebo treatment, a solution containing 9% (w/v) sodium chloride was used. 3 to 5 minutes before blood sampling, mice were anesthetized intraperitoneally with Mebumal® (pentobarbital) vet. 60 mg/ml. The dose volume was approximately 1.2 ml/mouse. Under anesthesia, 0.45 ml of blood was withdrawn from the vena cava into plastic syringes containing 0.05 ml of sodium citrate (0.13 M). Plasma was prepared from the blood by centrifugation (8800 g for 7 minutes) and then stored in Cryoflex plastic tubes at -70ºC until determination of VIII:C.

Results TABLE 3

VIII:C in plasma from mice given subcutaneous plasma-derived factor VIII, 10,000 IU/kg. The VIII:C in the administration solution was approximately 250 IU/ml.

Results

1. The bioavailability of plasma-derived factor VIII is quite low according to the results of the in vivo study in mice (Table 3). The bioavailability of plasma-derived factor VIII is approximately 1/10 of the bioavailability of r-VIII SQ. Thus, the bioavailability of r-VIII SQ is significantly higher than the bioavailability of human plasma-derived factor VIII. The maximum plasma level of VIII:C is seen approximately 1.5 hours after subcutaneous administration.

EXAMPLE 3

Recombinant factor VIII was prepared according to the procedure described in Experimental Section, with the following exceptions: (i) the material of r-VIII SQ obtained from the last purification step was diluted with a buffer not containing PEG, (ii) the r-VIII SQ solution was not lyophilized, it was stored at -70ºC.

The r-VIII SQ solution had the following composition:

Composition per bottle:

L-Histidine, mg 7.5

Sucrose, mg 158

Sodium chloride, mg 45

Calcium chloride (2H2 O), mg 1.25

Polyoxyethylene (20) sorbitan monooleate

(Tween 80®), mg 0.50

VIILC filled, IU/vial 6070

VIII:C, IU/ml* 1130

* Diluted r-VIII SQ solution (1 part r-VIII SQ solution + 1 part water, v : v).

The r-VIII SQ solution was diluted with sterile water for injection (1 part of r-VIII SQ solution + 1 part water, v:v) prior to administration when the dose of VIII:C was below 3000 IU/kg body weight.

Female cynomolgus monkeys (Macaca fascicularis) weighing approximately 3-3.5 kg were injected subcutaneously with the r-VIII SQ solution in the dorsal region. Depending on the dose, the injection volume varied between approximately 0.2 to 2.0 ml/kg body weight. Subcutaneous injections with single doses of 250, 2500 and 5000 IU/kg were administered. At each sampling, 1.8 ml of blood was collected into tubes containing citrate as anticoagulant (0.2 ml). After centrifugation, plasma was separated and frozen in aliquots (< -60ºC).

Results TABLE 4

VIII:C in plasma from monkeys receiving r-VIII SQ, 250 IU/kg, subcutaneously. The VIII:C in the administration solution was approximately 1130 IU/ml.

TABLE 5

VIII:C in plasma from monkeys receiving r-VIII SQ, 2500 IU/kg, subcutaneously. The VIII:C in the administration solution was approximately 1130 IU/ml.

TABLE 6

VIII:C in plasma from monkeys receiving r-VIII SQ, 5000 IU/kg, subcutaneously. The VIII:C in the administration solution was approximately 2470 IU/ml.

TABLE 7

Dose-response in monkeys receiving r-VIII SQ subcutaneously.

Dose: administered dose of r-VIII SQ (IU/kg body weight).

Response: VIII:C in plasma (IU/ml), approximately 9 hours after administration.

Result

1. According to the results (Table 4-6), plasma concentration as a function of time follows the typical pattern for a drug absorbed after subcutaneous administration. The maximum concentration of VIII:C is seen approximately 9 hours after administration.

2. The dose-response relationship obtained (Table 7) provides further confirmation that there is absorption of VIII:C into the bloodstream following subcutaneous injection.

3. The bioavailability of r-VIII SQ after subcutaneous administration in monkeys was approximately 5 to 10%. Bioavailability was calculated from the area under the activity (VIII:C)-time curve. Bioavailability was essentially independent of the dose of r-VIII SQ.

Claims (11)

1. A pharmaceutical formulation suitable for subcutaneous, intramuscular or intradermal administration which comprises highly purified recombinant coagulation factor VIII in a concentration of at least 1,000 ID/ml. 2. The formulation according to claim 1, which is designed for subcutaneous administration. 3. The formulation according to claim 1 or 2, in which the activity of factor VIII is more than 1,500 IU/ml. 4. The formulation according to any one of claims 1 to 3, which has a volume of 0.1 to 2 ml. 5. The formulation according to any one of claims 1 to 4, in which the factor VIII is a deletion derivative of recombinant coagulation factor VIII lacking all or part of the B domain. 6. The formulation of claim 1, which comprises: (i) at least 1,500 ID/ml of a deletion derivative of recombinant factor VIII lacking all or part of the B domain; (ii) at least 0.01 mg/ml of a polyoxyethylene sorbitan fatty acid ester; (iii) sodium chloride; (iv) a calcium salt; and (v) an amino acid in an amount exceeding 1 mM. 7. The formulation according to claim 5 or 6, wherein the deletion derivative of Factor VIII is the deletion derivative designated "Factor VIII SQ". 8. The formulation according to claim 7, in which the factor VIII SQ has been chemically modified by pegylation or covalently linked carbohydrates or polypeptides. 9. The formulation according to any one of claims 1 to 8, which is a stable aqueous solution ready for administration. 10. The formulation according to any one of claims 1 to 8, which is lyophilized and reconstituted with sterile water or a pharmaceutically acceptable buffer solution prior to administration. 11. The formulation according to any one of claims 1 to 8, which is mixed or reconstituted with an aqueous solution containing absorption enhancers or protease inhibitors prior to administration.